Hematology control standard comprising washed red blood cells and synthetic latex particles

ABSTRACT

A HERMATOLOGY CONTROL STANDARD FOR THE CALIBRATION OF BLOOD CELL COUNTING APPARATUS AND A METHOD FOR THE PREPARATION THEREOF BY ADMIXING PREDETERMINED AMOUNTS OF WASHED RED BLOOD CELLS AND SYNTHETIC LATEX PARTICLES HAVING A 5 TO 15 MICRON PARTICLE SIZE RANGE IN A FLUID SUSPENSION OF A KNOWN AMOUNT OF SERUM ALBUMIN.

United States Patent Olhce 3,558,522 Patented Jan. 26, 1971 US. Cl.252-408 6 Claims ABSTRACT OF THE DISCLOSURE A hematology controlstandard for the calibration of blood cell counting apparatus and amethod for the preparation thereof by admixing predetermined amounts ofwashed red blood cells and synthetic latex particles having a to 15micron particle size range in a fluid suspension of a known amount ofserum albumin.

This invention relates to a control standard for use in the counting ofblood cell particles. More particularly, this invention relates to acomposite and unitary hematological control standard for calibrating andchecking the accuracy of automatic or manual red blood cell and whiteblood cell counts, hematocrit and hemoglobin determinations.

Quality control has long been a necessary and routine procedure inclinical chemistry and coagulation laboratories. Accuracy in thecounting of red and white blood cells and in the making of hematocritand hemoglobin determinations of the patients serum is dependent, inpart, upon the use of adequate control standards. Thus, the accuracy ofthe manual technic of particle counting, such as by the classical methodof microscopy, can be checked by giving the technician a so-called blindsample, or control standard, containing a known concentration ofparticles for comparison with the unknown samples for which he is tomake a determination.

Modern technology has provided numerous types of automatic equipment forparticle counting which is gradually replacing the older and morelaborious manual techniques. But even these automatic methods ofparticle counting require constant quality control by the use of controlstandards since the possibility of malfunctioning of the instrument isever present. Consequently, the importance of accurate and reliablechecks on hematological determinations that may be used in the diagnosisof disease speaks for itself and needs no further amplification here.

The traditional method of calibrating automatic particle countingequipment has consisted of providing a whole blood standard byrepeatedly counting such blood by manual hemacytometer techniques toestablish its value.

The disadvantage of this method is that the standard is usable for onlyone day and each time a fresh whole blood standard is prepared, themanual counts must be repeated.

Other conventional methods consist of providing a red blood cellstandard in which the red cells have been stabilized by one means oranother to prolong their shelf life. However, these methods are notusable for white cell counts in a system which provides for thedestruction of the red cells by a lysing agent prior to counting of thewhite cells.

Still other methods consist of substituting simulated cell particles insuspension, for example, polystyrene latex particles, in place ofnatural blood cells. These methods have not been very satisfactory,however, since it has been too difficult heretofore to suitablysuspended sulficient particles in the carrier liquid.

Various other approaches to the problem of providing control standardsfor blood cell particle counting are described in recent US. Pats.3,406,121 and 3,412,037, and in recent British Pats. 1,129,873 and1,131,690.

It is an object of the present invention to provide a new and improvedcontrol standard for use in the counting of blood cell particles.

It is another object of the present invention to provide a composite andunitary hematological control standard for calibrating and checking theaccuracy of automatic or manual red blood cell counts, white blood cellcounts, hematocrit and hemoglobin determinations.

These and other objects will be apparent to the person skilled in theart after reading the disclosure hereof.

Briefly stated, the objects of the present invention are achieved byproviding a hematological control standard which comprises a fluidsuspension of a known amount of serum albumin containing predeterminedamounts of washed red blood cells and synthetic latex particles having aparticle size ranging from about 5 to about 15 microns, said fluidsuspension having a specific gravity and viscosity similar to normalblood serum.

The hematological control standard of this invention provides users ofautomated instruments with control for red blood cell counts, whiteblood cell counts, hematocrit and hemoglobin determinations all in asingle product, thereby eliminating the need for separate controlstandards for red blood cell counts and white blood cell counts as usedheretofore. The control standard has a shelf life of at least fourWeeks, which can be extended by fixing or stabilizing the viable redcells by known methods. It has been found that in this control standardthere is only about a one percent hematocrit decrease per week, due tothe natural shrinkage of the red blood cells, and virtually nohemolysis.

The synthetic latex particles used in the control standard of thisinvention are generally spherical in shape, they have a relativelyuniform size of from about 5 to about 15 microns, which approximates therelative size of the normal leukocytes, or White cells, and arepreferably employed in the fluid suspension at a concentration of fromabout 5,000 to about 10,000 particles per cubic millimeter,, which isthe approximate count in normal blood.

These latex particles can comprise polystyrene, polyvinyltoluene and/orstyrene-divinylbenzene copolymer latex and the like synthetic polymericlatex materials of suitable particle size.

The styrene-divinylbenzene copolymer latex particles are preferred foruse in this invention. These latex particles are visible under themicroscope under conventional magnifications at 10X and 40x, they areinert to the usual red cell lysing agents, such as acetic acid andvarious detergent substances, and otherwise provide suitable simulationof the white blood cells in the control standard of this invention.

The washed red blood cells are preferably employed in the controlstandard at a concentration of from about 3 million to about 5 millionper cubic millimeter, which approximates the count in normal blood.These washed cells are preferably obtained from 1 to 24 day old humanwhole blood, although any fresh red cells or viable red cells which havenot been lysed can be used. Careful and thorough washing of the bloodcells after separation from the plasma, such as by saline washing and/or washing in Alsevers solution, followed by filtering to remove anyresidual white blood cells, is desirable. As used herein, the termwashed red blood cells refers to red blood cells which have beenseparated from the other constituents of whole blood by the abovemethods or by any other conventional method of separating red bloodcells from contaminating substances.

The serum albumin is employed in the control standard of this inventionto provide a proteinaceous medium closely resembling plasma inconsistency. Serum albumin also has been found to provide a suitablemedium for suspension of the latex particles in the control standard ofthis invention without necessity of employing any of the other normalserum proteins. The serum albumin can be obtained from whole plasma byalcohol fractionation, ammonium sulfate precipitation, and any othersuch conventional procedures for preparing serum albumin.

From about 2 percent to about 5 percent, and preferably about 3 percent,by Weight of albumin is generally contained in the control standardfluid suspension of this invention. When the washed red cells and latexparticles, at approximately the normal particle counts of red and whitecells as herein'before stated, are suspended in an aqueous solution ofthe albumin at a pH of about 6, the control standard fluid suspension isfound to very closely resemble normal blood serum in specific gravityand viscosity.

It is preferable to additionally include in the control standard a smallbut effective amount of antibiotic or preservative, for example, Terramycin, neomycin, sodium azide, and the like antibiotics orpreservatives, for their biocidal effects. About one percent by weightof antibiotic or preservative in the final product is suitable for thispurpose.

The hematological control standard of this invention can be used forboth automatic and manual particle counting techniques as hereinbeforestated. The automatic equipment to which the control standard is adaptedis of great diversity. In general, one or the other of two types ofparticle size analysis equipment is employed. In one type, each particleis counted and its discrimination property is measured directly. In theother type, the particles are measured in bulk and particle behavior isrecorded through a series of measurements of the magnitude of the bulk,in terms of the count, combined surface area or combined mass. The typeof measurement used then determines the basis of the size distribution,

Optical and electrical properties are two of the most prevalent types ofsize discriminating properties employed in the particle size analysisequipment to which the control standard of this invention is adapted.The optical equipment can employ imaging, spectral transmission,scattering and diffraction mechanisms, while the electrical equipmentcan employ resistance, capacitance, and charge mechanisms.

A specific suitable example of an instrument useful with the controlstandard of this invention is the Coulter Electronic Blood Cell Counterand similar such equip ment as described, for example, in US. Pats.2,656,508, 2,869,078, 2,985,830 and 3,340,470. This particular instrument discriminates among particles by how they affect the electricalresistance of the fluid medium containing the particles in suspension asthey pass through an orifice.

Other examples of apparatus which can be calibrated by the controlstandard of this invention are the Technicon Instruments Auto Analyzersand similar such equipment as described, for example, in US. applicationSer. No. 427,593, filed Jan. 25, 1965. This apparatus provides a supportfor a plurality of whole blood samples which are sequentially diluted, aflow cell of small crosssection through which the volume of dilutedblood ispassed, illuminated optical means coupled to the flow cell fordetecting the passage of individual cells therethrough and providing anoutput pulse signal in response thereto, and electronic means forreceiving and totaling the number of pulses per unit of time andproviding an output signal.

Various other conventional types of particle counting instruments whichemploy the metering of a sample of the particle-containing suspensionpast a scanning point in the detecting system will be apparent to theperson skilled in the art.

In the use of the control standard in calibrating or checking theaccuracy of the above or similar such equipment, the fluid suspension ismixed well prior to its use to ensure good dispersion and prevent theparticle sizes from being indicated too large and size distribution toowide. The consistency of the fluid suspension of the control standard ofthis invention as described hereinbefore is capable of retaining theparticles in suspension without appreciable loss of particles bysettlement during the usual calibration procedures on equipment such asdescribed herein. Excellent calibration can be achieved when every fifthsample used in these instruments is the control standard of thisinvention. The employment of this control standard in place of anunknown blood sample at other predetermined periodic and regularintervals will similarly provide suitable calibration of various otherinstruments.

After making the cell counts, calculation of the red blood cell indicescan be made by the following formulas:

(a) Mean corpuscular volume (MCV) is the average of the individual redblood cell.

Hematocrit (percent) X 10 Red blood cell count (in millions) =MCV (incubic microns) (b) Mean corpuscular hemoglobin (MCH) is the averageweight of hemoglobin in the individual red blood cell.

Hemoglobin (g./l00 ml.) X 10 Redbloodcellcount (in millions) =MCH (inmicrograms) (c) Mean corpuscular hemoglobin concentration (MCHC) is thepercent of hemoglobin in the average red cell.

Hemoglobin (g./ ml.) X 100 Hematocrit (percent) The following examplefurther illustrates the invention herein although the invention is notlimited to this specific example. All parts and percentages herein areby weight unless otherwise specifically stated.

EXAMPLE Whole human blood containing anticoagulant is centrifuged andthe supernatant plasma is aspirated. Saline (an aqueous solution of 1.2%NaCl) is added to the packed cells in an amount sufiicient to replacethe volume of separated plasma. The packed cells are thoroughly mixedwith saline and centrifuged again. This saline washing andcentrifugation is repeated two more times.

The packed cells are then similarly washed three times with a modifiedAlsevers solution, pH 7.0, made-up by dissolving in one liter of Water,20.5 grams of anhydrous dextrose, 8.0 grams of sodium citrate-211 0, 4.2grams of sodium chloride and 5.2 ml. of an aqueous one percent citricacid solution. After the third washing with the modified Alseverssolutions, the red cells are again spun down and the supernatant isextracted.

About 40-50% by volume of an aqueous solution of 6 percent by weighthuman serum albumin in modified Alsevers solution (as described above)is then added to the washed red blood cells. The fluid suspension isthen adjusted to a concentration of about 5 million red cells per cubicmillimeter and 3 percent by weight of albumin.

Latex particles of styrene-divinylbenzene copolymer having a particlesize range of 6 to 14 microns and an average particle size of 7.6microns are then added to a count of about 10,000 particles per cubicmillimeter. The final product is then filled into bottles of 10, 20 and50 ml. size and is ready for use in calibration and checking theaccuracy of automatic and manual blood cell counting instruments asdescribed hereinbefore. The specific gravity of the final product issimilar to that of normal blood .serum (about 1.03) and its viscosity,as determined by its flow and other handling characteristics, veryclosely resembles that of normal blood serum.

= MCHO (in percent) Various other examples and modifications of theforegoing example will be apparent to the person skilled in the artafter reading the invention described herein and defined in the appendedclaims without departing from the spirit and scope of the invention. Allsuch further examples and modifications of the foregoing example areincluded Within the scope of the invention.

What is claimed is:

1. A fluid suspension of a known amount of serum albumin containingpredetermined amounts of washed red blood cells and synthetic latexparticles having a particle size ranging from about 5 to 15 microns,said fluid suspension having a specific gravity and viscosity similar tonormal blood serum,

2. The fluid suspension of claim 1 in which the concentration of serumalbumin is from about 2 percent to about 5 percent by weight, the redblood cell count is from about 3 million to about 5 million per cubicmillimeter and the latex particle count is from about 5,000 to about10,000 per cubic millimeter.

3. The method of making a hematological control standard for thecalibration of blood cell counting apparatus comprising the steps ofadmixing predetermined amounts of washed red blood cells and syntheticlatex particles having a particle size range of from about 5 to about 15microns in a fluid suspension of a known amount of serum albumin.

4. The method of claim 3 in which the concentration of serum albumin inthe fluid suspension is from about 2 percent to about 5 percent byweight, the red blood cell count is from about 3 million to about 5million per cubic millimeter and the latex particle count is from about5,000 to about 10,000 per cubic millimeter.

5. The method of calibrating automatic blood cell counting apparatuscomprising substituting the fluid suspension of claim 1 in place of anunknown blood sample in said apparatus at predetermined periodic andregular intervals. 6. The method of claim 5 in which the concentrationof serum albumin in the fluid suspension is from about 2 percent toabout 5 percent by weight, the red blood cell count is from about 3million to about 5 million per cubic millimeter and the latex particlecount is from about 5,000 to about 10,000 per cubic millimeter.

References Cited UNITED STATES PATENTS 3,406,121 10/1968 Jones 23-230X3,412,037 11/1968 Gochman et al 252-408 3,436,187 4/1969 Ferro et a1252408X 3,466,249 9/ 1969 Anderson 23230X FOREIGN PATENTS 690,849 7/1964Canada 252-408 HAROLD ANSHER, Primary Examiner J. C. GIL, AssistantExaminer US. Cl. X.R.

